Further purification of acetyl CoA carboxylase kinase -Purification of this enzyme is essential to understanding the regulatory mechanisms involving phosphorylation and dephosphorylation of key enzymes. Purification of acetyl CoA Carboxylase - There are some indications that cgnventionally purified carboxylase has already experienced a partial proteolysis (even in the subunit with a molecular weight of 230,000). Different purification procedures, using an AMP affinity column, have been investigated in our preliminary experiments. Such techniques provide much less chance for proteolysis than the conventional methods. Our purification attempts are also important in demonstrating the existence of two different phosphorylation sites on the enzyme. Peptide sequencing around the phosphorylation sites will require a large amount of enzyme. Regulation of covalent modification of acetyl CoA carboxylase appears to be dependent on the adenylate energy charge system of phosphorylation. Our preliminary experiments indicate that the carboxylase has an additional ATP binding site which is recognized by AMP. Further attempts to establish the existence and nature of the two ATP binding sites on the carboxylase by affinity labelling and equilibrium dialysis technique and to determine how phosphorylation is regulated will be carried out. Relationship between phosphorylation and disaggregation of acetyl CoA carboxylase - Phosphorylation of the carboxylase causes disaggregation of the enzyme. Using ultracentrifugation and 32P labelling, we hope to relate these two events to the inactivation of the enzyme under different physiological conditions.